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Conformational Study in Water by NMR and Molecular Modeling of Cyclic Glutamic Acid Analogues as Probes of Vitamin K Dependent Carboxylase Vale´ry Larue,† Josyane Gharbi-Benarous,†,‡ Francine Archer,† Robert Azerad,† and Jean-Pierre Girault*,† Universite´ Rene´ Descartes-Paris V, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (URA 400 CNRS), 45 rue des Saint-Pe`res, 75270 Paris Cedex 06, France, and Universite´ Denis Diderot-Paris VII, UFR Chimie, 2 Place Jussieu, F-75251 Paris Cedex 05, France Received October 10, 1995X
The conformational analysis of four glutamic acid analogues containing a cyclopentyl or cyclohexyl ring, substituted in position 1 by a BOC protected amino group and a methyl ester group and in position 3 by a free carboxylate group, has been carried out in an aqueous environment, by 1H and 13C NMR spectroscopy, and molecular dynamics (MD). Their structural properties were under investigation for a structure-activity relationship analysis to determine the preferred conformation in the carboxylase active site. For each compounds, resulting conformations from NMR and MD data were analyzed and classified according to the dihedral angles χ1 and χ2, the distances and the spatial distribution involving charged or substituted Cand N-terminal groups. A reduced number of conformational families were found to be in qualitative agreement with NMR and MD data. A comparison between these different classes of the active and nonactive derivatives was achieved. A liver microsomal carboxylase is responsible for the vitamin K-dependent conversion of selected glutamic acid residues of precursor proteins to γ-carboxyglutamic acid (Gla) residues of several plasma clotting factors1-3 (the historical aspects of vitamin K action have been adequately reviewed4) (Figure 1). Using relatively simple substrate analogues such as the tripeptides BOC-Glu-Glu-Val corresponding to the N-terminal sequence of descarboxyprothrombin or shorter substrates, BOC-Glu-OMe, it was demonstrated that the decarboxylation reaction occurred.5-9 These lowaffinity peptides have proven to be useful for demonstrating several aspects of the mechanistic features of the reaction.5-12 In the search for more efficient carboxylase inhibitors, it was necessary to rationalize structurally-related affinities, that might result from privileged interactions of the side chain carboxylate group with the carboxylase active site and possibly to determine the conformation of the Glu side chain in the inhibitor-carboxylase complex. The cyclic glutamic acid analogues BOC-X-OMe where X was conformationally restricted glutamic acid analogues 1-5 (Figure 2), such as the cyclo derivatives, 1-amino-1,3-dicarboxycyclohexane or -cyclopentane (cis or trans), were tested13 with the intention of directing the carboxylic group of the side chain in a definite position, allowing a three-dimensional exploration of the binding site of the carboxylase5-9 (Table 1). We report here a NMR and MD study in aqueous solution, for the cisand trans-1-[(tert-butyloxycarbonyl)amino]cyclohexane(or cyclopentane)-3-carboxylic acid 1-methyl ester, hereafter named respectively, BOC-cis-C6-OMe 6 and trans-7, BOCcis-C5-OMe 8 and trans-9 (Figure 2), in order to understand their structure-activity relationship in evaluating the spatial position of the γ-carboxylate of the side chain relative to the peptide chain. †
Universite´ Rene´ Descartes-Paris V. Universite´ Denis Diderot-Paris VII. X Abstract published in AdVance ACS Abstracts, April 15, 1996. ‡
S0095-2338(95)00153-3 CCC: $12.00
Figure 1. The liver microsomal vitamin K-dependent carboxylase. The enzymatic carboxylation reaction is coupled to the oxidation of the reduced hydronaphtoquinone form of the vitamin to the vitamin K-2S,3R-(+)-epoxide. The 3-position is occupied by a phytyl group in phylloquinone and by an unsaturated polyisoprenyl group of varying length in the bacterially synthesized menaquinones. The stereochemistry of the reaction has now been solved and the γ-pro-S hydrogen of the glutamyl residue is removed followed by inversion of configuration during CO2 addition.
RESULTS AND DISCUSSION
Conformational Analysis. These derivatives display some flexibility. In cyclopentane the angle of maximum puckering rotates without substantial change in potential energy. However, the presence of the three substituents will give rise to an induced potential energy barrier opposing free pseudorotation. The consequence of “limited” pseudorotation and a description of conformations was developed. In the present article, we propose an accurate description of rings in cyclohexyl and cyclopentyl analogues and the correlated conformations of the C(3)-(γ)CO2H side chain. The methodology presented illustrates the usefulness of molecular modeling in elucidating possible solution confor© 1996 American Chemical Society
718 J. Chem. Inf. Comput. Sci., Vol. 36, No. 4, 1996
LARUE
ET AL.
Figure 3. Pseudorotational pathway of cyclopentane ring for (a) Boc-cis-C5-OMe 8 and (b) Boc-trans-C5-OMe 9. Each exterminal point of dotted radials in the circle represents a specific E form or T conformations. Heavy arrows indicate the corresponding family type.
Figure 2. (a) Structures of R-substituted and cyclic glutamic acid analogues 1-5. (b) Structures of cyclohexane and cyclopentanederived analogues of glutamic acid: cis- and trans-1-[(tertbutyloxycarbonyl)amino]cyclohexane (or cyclopentane)-3-carboxylic acid-1-methyl ester, named respectively, BOC-cis-6 and transC6-OMe 7, BOC-cis-8, and trans-C5-OMe 9. Table 1. Carboxylation of the Tripeptides Boc-X-Glu-Val or the Derivatives Boc-X-OMe (X ) Glutamic Acid Analogues) BocX-Glu-Val or BocX-OMe
Km (mM)
Vmax Ki nM CO2 incorporated (mM) mn-1 (mg protein)-1
Boc-Glu-Glu-Val 1.7 ( 0.2 Boc-(DL)-2Me-Glu-Glu-Val 18.5 ( 3.5 Boc-(DL)-cis-C6-Glu-Val 15.5 ( 1.5 Boc-(DL)-trans-C6-Glu-Val 7.0 ( 1.9 Boc-(DL)-cis-C5-Glu-Val 12.2 ( 0.8 Boc-(DL)-trans-C5-Glu-Val 4.4 ( 0.0 Boc-Glu-OMe 5.5 ( 0.5 Boc-(DL)-2Me-Glu-OMe >60(b) Boc-(dl)-cis-C6-OMe 65(b) Boc-(dl)-trans-C6-OMe 45(b) Boc-(dl)-cis-C5-OMe 40(b) Boc-(dl)-trans-C5-OMe 20(b) b
0.93 ( 0.01 0.50 ( 0.04 0.80 ( 0.10 0.35 ( 0.05 0.80 ( 0.08 0.40 ( 0.05 0.34 ( 0.01
a Competitive inhibition relative to the substrate Boc-Glu-Glu-Val. Competitive inhibition relative to the substrate Boc-Gln-OMe.
mations and to explore fully the conformational space of these compounds and NMR data in revealing that approximate solution structures can be estimated. NMR parameters reflect the virtual conformation and at the same time the structural information of the different conformations generated by MD are of great benefit in predicting the conformation in solution. The different biological activity observed in the formation of an inhibitor-carboxylase complex could be due to structural difference, localized flexibility, minor conformers, or a particular transition state implied in reaction with the receptor. The preferred conformer of cyclohexane is the chair form in which all dihedral angles have an absolute value close to 56°. In this conformation all bonds (endocyclic as well as exocyclic) are staggered thus explaining the minimum in conformational strain. The energy barrier to convert a chair
form into another one is close to 10-12 kcal mol-1; this chair-chair conversion is a complex process of cycle inversion.14 The chair form of cyclohexane is considered “rigid” since it must undergo cycle inversion of relatively high energy to yield other conformers. However besides the rigid chair conformers, cyclohexane also exists as flexible forms which include the boat conformer and the twist form. The boat and twist forms represent the local energy maxima with the former about 5-6 kcal mol-1, above the global minimum of the chair forms. The boat and the chair forms show only equatorial and axial substituents positions. In cyclopentane the bond angles have values close to the optimum; therefore, the strain in the molecule arises essentially from bond opposition and is partly relieved by puckered conformations. In the two flexible forms, enVelope E and halfhchair T conformers, the following types of exocyclic bonds have been recognized: equatorial, axial, and isoclinal or bisectional bonds for ring substituent positions. The two conformers E and T are extremes of symmetry in the pseudorotational circuit of cyclopentane: 10 envelope and 10 half-chair forms interconvert by this process (Figure 3). The pseudorotation circuit of cyclopentane is essentially of constant strain without substituent; in contrast the trisubstituted cyclopentanes 8 and 9 present maxima and minima with the less stable conformer about 4 kcal mol-1 above the global minimum.14-17 The final structures obtained after several mechanics molecular calculations were examined for the overall energetic favorability and compared with the structure derived from the NMR data. The torsion angles of generated structures can be correlated to the corresponding coupling constants by using Karplus-type equations.18-20 NMR Spectroscopy. For the four derivatives, only one set of proton and carbon resonances was observed, except for the N-BOC and ester chain which indicates that slow conformational change occurs for these two parts of side chains. For the cyclic residue, a unique set of resonance lines was found for each atom. This could result from an averaging of chemical shifts and coupling constants due to fast conformational changes. The conformational analysis is based on the vicinal 3J1H,1H and 3J13C,1H coupling constants reported in Table 2. Determination of heteronuclear 13C,1H coupling constants by the selective 2D INEPT experiment21 is especially helpful for 3-H bonds in order to determine the relative positions of the substituents on the C5-ring. The vicinal coupling constants were obtained from these spectra by iterative techniques on a spectral simulation (PANIC). The BOC-cis-C6-OMe 6 always adopts the chair conformation C1 with γ-CO2- equatorial and the minimal sterical
CYCLIC GLUTAMIC ACID ANALOGUES
J. Chem. Inf. Comput. Sci., Vol. 36, No. 4, 1996 719
Table 2. 1H, 1H and 1H, 13C Coupling Constants in D2O (Hz, Error 0.5 Hz) Values Confirmed by Simulation of the Signals (Program PANIC)a J/Hz
6
7
8
9
J
2a,2b 4a,4b 5a,5b 6a,6b
-12.5 -12.5 -12.5 -12.7
-12.7 -12.8 -13 -12.9
-13.5 -12 -12
-13.5 -12 -12
J
3,2a 3,2b 3,4a 3,4b 4a,5a 4a,5b 4b,5a 4b,5b 5a,6a 5a,6b 5b,6a 5b,6b
12.8 3.5 12.8 3.2 3.5 12.5 3.4 3.2 3.4 3.5 12.7 3.5
3.8 10.8 3.8 10.3 4.0 4.5 11 4.0 4.0 11 4.0 4.0
7.5 8 7.5 9 9.5 6 5 8
2
3
3J
3-H,1-C 3-H,5-C
